Comparison of various diets in the study of Achromotrichia and growth of rats

A COMPARISON OP VARIOUS DIETS . IN THE STUDY
OP ACKROMOTRICHIA AND GROWTH OP RATS

by

JAMES KELIJf WOODS

B, S,, Kansas State College of Agriculture
and Applied Science, 1939

A THESIS

Submitted in partial fulfillment of the
requirements for the degree of

MASTER OP SCIENCE

Departnient of Chemistry

KANSAS STATE COLLEGE
OP AGRICULTURE AND APPLIED SCIENCE

1942

KANSAS STATE COLLEGZ LIBi^ARIES

Ijocv-
LO

Ml

TABLE OP CONTENTS

Page

INTRODaCTION 2

REVIEW OF LITERATURE 3

EXPERIMENTAL PROCEDURE 11

RESULTS AUD CONCLUSIONS 18

Growth « • 18

Achromotrichla.. 21

SUMMARY 27

ACKNOWLEDGMENT 28

LITERATURE CITED 29

2

INTRODUCTION

The description of the syndromes resulting from the
deficiencies of the individual vitamins in the diets of
rata make up a very important part of tho study of these
dietary factors, Howover, variations in ooaervationa,
basal rations, methods of feeding and care, and uncontroll-
able factors such as the differences in the vltnmln require-
ments of different strains of rats have led to considerable
confusion in the llterat^ire.

In the entire field of vitamin deficiencies affecting
the condition of the skin and the quality and color of the
hair, several factors including panthothenic acid, pyrldoxine,
certiln of the fatty acids, and p-aminooenzoic acid appear
to be involved. The interrelationships existing between
these factors and the influence of other vitamins and feed
ingredients have not been adequately studied.

Grey hair was observed in rata fed on a bread and
whole milk diet as early as 1923 (17), It was later shown
by Gorter (14) that this type of greynesa was due to a
deficiency of copper in the diet. In 1938, Morgan et al
(30) reported a greying of black rata in which copper had
no curative effect.

An attempt to find the specific factors involved in
the cure of this achromotrichia (grey hair) has opened

3

a new field for Investigation. Several confllctin,. reports
regarding the factors involved have already made their
appearance. Henderson, et al 0.9) have reported that pan-
tothenic acid will prevent or cure grey hair in black rats.
Anabacher (1), on the other hand, has found that in black
rata fed a liberal quantity of this vitamin, an achromo-
trichia develops which can be cured by the administration
of p«aminobenzoic acid.

The diets used by Henderson, et al and Anabacher
differed in several respects in quality and quantity of
fat and vitamins. If the differences in the results re-
ported had a real nutritional basis, then It was probably
due to the differences in quality and quantity of fat and
vitamins fed because otherwise the diets were practically
the same .

The purpose of this work was to study the effect of
Interchanging soim of the components of these two diets
on growth and achromotrichia with ulack rats as the
experimental subjects.

REVIEW OF LITERATURE

Aa early as 1930, TTorrls and Ringrose (33) described
a pellagra-like syndrome in chicks. By 1936, thiamin and
riboflavin were available in crystalline form and the
symptoms resulting from their absence in the diet had been
fairly well characterized. It was known that they could

be readily removed from aqueous rice bran or liver extracts
by a relatively small amount of fuller's earth, Lepkovsky,
Jukes, and i^rause (2P) found that the filtrate from the
above absorption could be treated with more fuller's earth
to absorb a Factor I which proved to be vitamin Bg or py-
ridoxine. The filtrate still contained a PV-ctor II, or the
filtrate factor. The filtrate factor prevented or c\ired
the chick dermatitis, and both factors were essential for
rat growth*

In 1938, Williams et al (48) separated pantothenic
acid, a yeast growth factor, from liver. Jukes (20) and
Woolley, Waisraan, and Elvehjem (52), working with samples
furnished by Williams, fotind it to be identical with the
filtrate (chick antiderma title) factor. Unna (44), in
the following year, described the pantothenic acid deficiency
symptoms of the rat. He found 80 y per rat per day to be
the approximate maintenance dose for optimal growth.

In 1923, Hartwell (17) reported a greying in rats diiring
hot weather on a bread and whole milk diet. He attributed
this greying to the lack of tryptophane and tyrosine for
melanin or black pigment formation. But Keil and Nelson
(21) showed copper to be the curative factor for greying
in rats on a whole milk diet, Gorter (13) produced a yellow-
ish depigmentation on a synthetic diet. After trying many
vitamins and minerals, copper was again found to be the only
active principle for prevention or cure (14).

5

In 1938, Luanda and Krlngstad (23) and Morgan, Cook,
and Davison (30) reported greylnp; In piebald and black
rata respectively. Morgan et al reported copper to have
no curative effect. Liuide and Krlngstad (?4) concluded,
the following year, that the growth- promoting filtrate fact-
or was not identical with the anti-greying filtrate factor,
Morgan and Simins (31) stated that injections of commercial
adrenal cortex and thyroid extracts cxired the greying in
rats slowly but did not restore growth. They also found
that dogs and silver foxes became grey on the filtrate fact-
or free diet. Oleson, Elvehjem, anu uart (34) reported that
pantothenic acid concentrates were ineffective in preventing
the greying now becoming known as experimental nutritional
achromotrichia.

Free (11) settled the question of the existence of more
than one type of achromotrichia. He showed conclusively
that there are at least two types of greyneas in rata: that
resultini^ from a deficiency of iron, copper, and manganese
and accompanied by nutritional anemia, and that resulting
on a synthetic diet in which the hemoglobin level is normal,
and curable with rice bran but not with rice bran ash.

The reports on the Inefficacy of pantothenic acid and
the belief that the filtrate factor contained vitamins other
than pantothenic acid which cured nutritional achromotrichia
were made by several workers before pure pantothenic acid

6

was avallabla and before the pantothenic acid requirement
of the rat was known, Gyorgy and Poling {li>) found synthetic
pantothenic acid to have a curative action, Unna, Richards,
and Sampson (45) fed clack and piebald rata on a diet ade-
quate except for the absence of pantothenic acid. They ob-
served greying of the fur in three to seven weeks along
with retardation of growth, the appearance of scant coarse
fur, inflammation of the nose, "blood-caked" whiskers, and
adrenal hemmorrhages. Development of the achromotrichla and
other lesions in normal rats was prevented by the addition
of 100 y of Ca pantothenate per rat per day. It restored,
within three to four weeks, the black pigmentation to the
rats which had become grey. They found the ability of
liver and rice bran to cure achromotrichla to parallel
their pantothenic acid content although they exerted a
growth-promoting effect superior to that of pantothenic
acid« Blotln, inositol, and p-amlnobenzoic acid were found
to be ineffective, giving no differences in greying, growth
rate, or the presence of scattered grey hairs persisting
after addition of pantothenic acid to the diet, A 68 per-
cent level of sucrose and cere lose were used interchange-
ably in their diets as were eight percent levels of hydro-
genated vegetable oil (Crisco) and butterfat without any
apparent differences in results. Henderson et al (19)
studied only pantothenic acid and p-amlnobenzolc acid
with similar results.

Mushott and Unna (32) fotmd the dally administration
of adrenal cortical extract, deaoxycorticoaterone acetate,
thyroid, and anterior pituitary extract to have no preven-
tive or curative action on greying of hair or occurrence of
adrenal hemorrhages in rata on pantothenic acid deficient
diets, McElroy et al (26) found the red deposit aroiind the
nose and on the whiskers of pantothenic acid deficient rata
to be coproporhyrln from the Harderlan gland and not blood
as it had been previously believed.

Not all workers have been able to maintain complete fur
color through the use of pure Ca pantothenate. vVilliams (47)
reported the failure of either pantothenic acid concentrate

or synthetic Ca pantothenate to prevent or cure rat achro-

n
motrlchia. Gyorgy and Poling (16) found blotin to be an

additional factor. Enwrson and Evans (9) found Ca pantothen-
ate to prevent pattern greying but not salt and pepper grey-
ing which they called stippling. They reported a liver fil-
trate to protect completely against greying.

In 1940, Woods (49) found p-aminobenzoic acid to have a
high activity in antagonizing sulfanilamide, Rubbo and
Gillespie (39) isolated N-benzoyl-p-aminobenzolc acid from
brewers' yeast and found It to be a growth factor for bacter-
ia, Ansbacher (1) reported p-amlnobenzoic acid to be a
vitamin the following/ year. He fed 500 r each of Ca panto-
thenate, inositol, and nicotinic acid, 3 mg of choline chlo-
ride, and 40 V each of thiamin, pyrldoxine, and riboflavin

8

in i ml of 20 percent ethanol solution to each rat dally in
addition to a synthetic basal diet. On the appearance of a
definite greying, a second daily supplement of 1 ml of 20
percent ethanol solution containing 3 »g of the p-aminoben-
zolo acid per milliliter was fed. A bluish dlacolorlzation
of the akin typical of the first sign of growth of normally
pigmented hair appeared in two to three weeks, l lack hair
appeared within a month,

Martin et al (29) reported p-amlnobenzolc acid to have
a modifyine; effect on melanin formation and pantothenic
acid to have none. It was also shown that p-aminooenzoic
acid prevented greying in mice on a high pantothenic acid
diet (28). Sure (43) showed It apparently to be a dietary
essential for lactation and reproduction In the rat. Sieve
(40) reported it as well as a vitamin 3 complex preparation
to be active in curing achromotrichia In humans, Martin
(27) reported that p-amlnobenzolc acid deficiency in rats
is characterized by slight greying. He further concluded
that the ratio of Ca pantothenate to p-amlnobenzoic acid
was the important factor, aal that a ratio favoring the
latter resulted In greying. He offered the explanation
that there is a destruction of pantothenic acid by micro-
organisms which are stimulated in their growth by p-amlnoben-
zolc acid. Ansoacher, vVisansky, and Martin (2) showed that
the acid probably has a protecting or sparing action on
certain hormones. Richards (37) found it to have a low
acute and chronic toxicity.

9

There have also been reports in which p-aminobenzoic
acid has been shown not to ne effective in connection with
achromotrichla and alopecia. The studies of Unna et al and
Henderson et al have already been cited. Emerson (8) failed
to get any curative response with p-aminobenzolc acid on rata
previously greyed on a pantothenic acid deficient diet. Woolley
(51) found it ineffective in the treatment of alopecia in mice,
Richter and Clisby (38) produced achromotrichla iu rats by
administering the drug phenylthlocarbamide, showing that a
positive factor, a poison, may cause greying as well as a
deficiency of an essential factor*

Inositol la one of the more recently reported members
of the vitamin B complex. In 1941, Pacvek and Baum (35)
reported inositol to be a growth- promoting factor for the
rat and to prevent the symptom known as "spectacle eye".
Woolley (50) demonstrated that the mouse required inositol
for normal growth and the prevention of alopecia. Pantothenic
acid was also found to be an anti-alopecia factor, and it
was also noted that many pantothenic acid deficient mice were
unable to open their eyes and showed a paralysis ;>i the hind
legs. Hegsted et al (18) stated that Inositol has a definite
growth- promo ting action in the chick although no other path-
ological symptoms had been produced by its lack.

In 1937, Elvehjem et al (7) found nicotinic acid and
nicotinamide highly effective in curing canine black tongue.
The following y«ar. Spies, Bean, and Stone (41) reported it

10

b«n«floial to both th« phyaloal and mental symptoms of pell-
agra* Madison, Miller « end Kaith {95) curad awina pallagra
with nicotinic acid, Dann and Kohn (5) reported that rats
wore able to synthenise nicotinic acid, and that the rata of
growth was not Increaaed by adding It to the diet. Their
findings indicated that nicotinic acid ia not a vitomin for
the rat. Stekel (4?) recently fo\ind one percent niacin (nio«»
otinic acid) to inhibit growth in yoiin^ male rats fed a caaein
low diet* Methionine or cystine promptly alleviated the in-
hibition, but neither choline nor glycine were effective. No
niacin inhibition was observed in the female* Hie observa-
tions also suggested that niacin was converted principally
into trigonelline in the male but not in the female, and tibiat
methionine or its labile methyl group is involved*

In 1935, r>est aad untSBun (3) proposecl that choline
be considered a dietary essential in the rat. Griffith (12)
stated that the daily requirement of choline varies with the
dietary methionine, cystine, betaine, and cholesterol as
well as with the adequacy of the ration for optimum t'^owth*
Choline-llke action of methionine and betaine appears satis-
factorily explained on the basis of transfer of methyl groups
by the process of transmethylation proposed by du Vigneaud*
Deuel et al (6) stated that rats previously on a high butter-
fat diet supplemented with choline did not accumulate fat in
the liver* *ngel and Salmon (10) produced fatal toxicity and
described the external and microscopic syrapt(»as of rats on a

11

choline deficient diet. The ayaiptoiaa were prevented by
feeding 20 to 30 mg of choline chloride per rat daily.
Emerson (8) reported that rats maintained on a 24 percent
casein diet apparently do not need choline.

EXPSRIidSIITAL PKOCEDURE

The purpose of this work was to study the growth and
achromotrichia produced in black rats on the Henderson,
et al diet (19), the Ansbacher diet (1) and six other
diets prepared by interchanging the different fats and
vitamins of the two diets.

Eight nearly equal groups of five 24-day-old black
rata each were used on the experimental diets. Each group
contained three males and two females . Ten rats, three
males and seven females, were fed the stock fox-chow diet
and used as positive controls. All the rats were kept in
individual cages set on coarse woven-wire trays. They were
given food and water ad libitum, and their weights and gains
in weight were recorded each week.

The eight experimental groups were designated alpha-
betically. Groups A to 1:, and they received Diets A to H
respectively. These rats were numbered consecutively one
to 40 beginning with the first rat in Group A and ending

The first group contained two males and three fe-
males.

12

with the la3t rat in Group H* The controls were numbered
from 40 to 50.

The experimental diets were prepared with the compo-
sitions shown in Tacle 1, Diet A was the Henderson et al
diet with sucrose replaced by cerelose. Diet B was the
Ansbacher diet with agar replaced by cellu flour, a diff-
erent salt mixture, and with the vitamins fed by mixing
into the feed instead of feeding separately in a PC per-
cent ethanol solution.

The six other diets were made up by interchanging the
cellu flour, soybean oil, Crisco, and cocl liver oil of the

Ansbacher diet for the corn oil and Natola of the Hender-

2
son, et al diet , and by adding different vitamin mixtures

to the diets. In this way it was hoped to find a "hidden"

factor or hidden factors which were responsible for the

differences in the results obtained by Ansbacher and the

Wisconsin workers.

The salt mixture used is shown in Table 2 and is
based on that of Phillips and Hart (36) with MnSO^ In-
creased as recommended by Conger and Elvehjem (4).

The casein used was extracted with alcohol. The
Natola which was used as a source of vitamins A and D,

2

The cerelose level was either raised or lowered to
make up for the difference in weight of corn oil and
Ansbacher 's fat -nixture plus cellu flour.

13

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15

Salts

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contained 55000 U.S. P. units of A and 5500 units of D
per gram.

In order to facilitate the mixing of the feed, the
salt mixture was dehydrated, and the amount used was re-
calculated to allow for the loss of water of hydration.

The casein and dehydrated salt mixture were ground
separately in the Wiley lill and mixed with ceroloso by
hand. Cellu flour was mixed in similarly. The fats were
mixed in by starting with a small addition of the diet to
the fat and adding more gradually until the fat was mixed

uniformly throughout the feed. The proper vltamlna were
mixed into the different diets by the same method used
for the fats. The choline was ground into a small amount
of ration in a mortar as a preliminary step to the aoove
procedure.

The rats were kept on the diets shown in Table 1
for six weeks. Diiring this time, only Groups A and C,
the only groups not receiving pantothenic acid, had de-
veloped achromotrichla. Their weights were significantly
lower than those of the other groups also. At the begin-
ning of the seventh week the diets of Groups A and C were
changed in an attempt to cure their achromotrichia. Diet
A was supplemented by mixing p-amlnobenzolc acid into the
diet at a level of 300 mg per 1000 g. Group C was shifted
to Diet B, the diet used by Ansbacher to produce greyness
although it contained a high level of pantothenic acid.

Two weeks later, the rats in the six other groups
had developed some scattered grey hairs or a slight salt
and pepper greyness, A method of judging the amount of
greyness In the eight groups was devised. Their growths
were also studied.

It was decided to supplement some of the diets at
this time in an attempt either to cure the slight salt and
pepper greyness or to produce a more definite achromotrichia.

IT

Diet H and part of Diet B were supplemented with 300 llg
of p-amlnobenzoic acid per 1000 g of feed. This made the
supplemented Diet B alriost Identical in composition to
the diet Anabacher used to cxire achromotrichia. The supple-
mented Diet B was fed to Group B, and in addition to rat
No« 15 of Group C which was entirely grey and to No. 2 of
Group A which was entirely grey ventrally out otherwise
black (symmetrical greyness). The rest of Group C which
contained rata Nos. 11 and 12 having the same type of
greyness as Noa. 15 and 4 respectively, were left on the
unsupplemented Diet B, If the two rats on the Diet B
supplemented with p-aminobenzoic acid were cured of grey-
ness while the two on the unsupplemented Diet B failed to
respond, it would be at least an indication of the benefi-
cial action of p-amlnobenzoic acid on achromotrichia.

Group A was not responding to p-amlnobenzoic acid in
the absence of pantothenic acid, so the diet was resupple-
n»nted to study the Importance of the pantothenic acid-
p-aminobenzoic acid ratio as suggested by lr!artin (27).
Ca pantothenate and p-aminobenzolc acid were added to
1000 g of Diet A at the levels of 10 mg and 500 mg respect-
ively. An even lower ratio of pantothenic acid to p-amino-
benzolc acid, or 10 mg Ca pantothenate and 1 g p-amino-
benzoic acid were added to 1000 g of Diet E, Nicotinic

18

ael<a «m« ftdd»d to Diet F at a oiia percent level to see if
its reported toxic effect on msle rets would have any ln»
fluence on their achromotrlohle*

OpoMp D WIS left on the unsupplemented Diet D to act
ss a control group. Group 0 was destroyed to conserve feed*

After 12 weeks, or four weeks after the above supple*
nentlng, the achromotriohla of all the gj»oups was again
studied*

. RBmVSB AHD COHCl-aSIOli

Growth

Table 3 clvos the av&vei^e weekly weights and average
total gains of the eight groups for the first eight weeks
of the experiittent. The slow growth of Groups A and C was
due to the absence of pantothenic acid in these diets*
?h« sharp increase in the rate of gain of Oroup C beginning
with the seventh week was due to the addition of pantothenic
acid to the diet* The p-aaiinotoensoio acid added to Diet
A at the same tlrae had no effect*

A comparison of the (^owth and dleta of the other
groups gave several indications of the growth- promoting
value of the fats and the vitamin mixtures which varied
from diet to diet*

19

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20

Interchanging cellu flotir, soybean oil, Crisco, and
cod liver oil of the Anabacher diet for corn oil and Natola
of the Henderson, et al diet seejned to have no effect. This
can be shown by comparing the average total (<alns of Group D,
receiving corxx oil, with Groups E, H, and D receiving the
"Ansbacher fats". These four groups received the Anabacher
(1) levels of thiamin, pyrldoxlne, riboflavin, and choline
while Group P received the Henderson, et al (19) levels,
and Group G received the Henderson et al levels of thiamin,
pyrodoxlne, and riboflavin, but no choline. The results
indicate that it would be safer to feed the Ansbacher
levels of thiamin, pyrldoxlne, ana riboflavin, if the op-
timum growth- promoting; effects of these three vitamins are
desired.

The fact that young rats on an 18 percent casein diet
need some choline Is well established, and the fact that
one member of Croup G died during the second week is in
agreement with this. Group E received 1 g choline per
1000 g of diet while Group B on an otherwise Identical
diet received only 300 mg choline per 1000 g. A coapar-
Ison of the weights of Groups B and E again showed that
feeding the higher level, this time the Henderson et al
level, would be safer*

Group H received no Inositol or nicotinic acid while
Groups E, D, and B on otherwise similar or Identical diets
received the Anabacher levels of these two factors. The

21

results In Taule 3 show that inositol and nicotinic acid
had no effect on growth,

Achronotrlchia

During the fourth week on the experimental diets, male
rats No, 2 and No. 11 on the pantothenic acid-free Diets A
and C respectively were definitely t'r»ylng. The greyness
appeared evenly distributed over the entire body except for
a black patch on the head. By the beginning of the fifth
week, females Mo. 5 of 'Iroup A and No. 15 of Group C were
greying in the same manner. By the sixth week, four rats
In each group showed some detiree of achromotrichia, Plate I
shows the difference in size and greyness of rat No, 11 of
Group C an-^ rat No. 21 of Group E on a diet containing pan-
tothenic acid. Rata "^o. 4 of Group A and '*'0. 12 of Group C
were just beginning to show a symmetrical greyness starting
ventrally and proceeding equally up both aides of the body.
The head of rat No, 12 was grey where bhe rats showintS the
more complete type of greyness had remained black, Pejnales
No, 3 of Group A and No. 14 of Groap C still were quite black.
Rats No. 1 of Group A and No, 16 of Group G had difficulty
opening their eyes. Moat of the rats exhioited ru3t-colored
hair on the nose and neck, tjrplcal of pantothenic add de-
ficiency in both albino and black rats.

Diets A and C had given almost Identical results al-
though Diet A contained corn oil while Diet C contained
the fats used by Ansbacher. This showed that the character

-^f'r

1»U»ATI0H OF PUTE I

Photographs of two r^ta thowini; tho effect of
diotfefy iMintdthdnic add on th# growth and aohroao*
trlchia of black rata after six waeics on axpariroantaX
diets* Tho rata ware of the same size aa". color at
the beginning of the experiment*

Fig* I« Showing' the totally black coat of rat
Ko* 9,1 which hod reoelved Diet B containing a liberal
quantity of pantothenic acid* The iprey sheen appe»r-
Ing in the ooat was due to the pliotography*

Fig* e» Shoving the gr«y ooat of rat "(O* 11 which
had received Diet C containing no pantothenic acid.

22a

PLATE I

Pig. 1. Fig. 2.

23

of the fats was not a significant factor in these diets.

The diets for Groups A and C were supplemented with
p-aninobenzoic acid and pantothenic acid I'eapectively, as
previously described, at the beginning of the seventh week.

By the naxt week, rat No, 1, still not receiving any
pantothenic acid, had developed the only typical "blood-
caked" whiskers observed, and his eye condition was worse.
The eye condition of rat No. 15 of Group C, had disappeared
promptly upon receiving the diet containing pantothenic acid.

At the end of eight weeks, all of the rats on the diets
which had contained pantothenic acid from the beginning show-
ed soiae salt and pepper greyneas. The control rats showed
this same condition, Tho rata from Croups A to H were scor-
ed for ^reyness by the method shown in Taule 4, and the in-
dividual and total scored obtained are shown in Table 5,
The rats in Groups A and C haa been on supplemented diets
for only two weeks which had been insufficient time for
any noticeable change in their greyness.

This method of scoring brought out two important
conclusions. The first conclusion was that none of the
diets protected the rats completely from achronotriohiaj
the second conclusion was that the nmles were more suscept-
ible to greying on these diets than were the females.

Starting with the ninth week the supplemented diets
described on page 16 were fed. After four weeks on these

S4

Table 4# Method of scoring the degree of greyneaa
of black rata.

Explanation of scoriae:

0 — Black

1 — Very slight salt and popper on sides

2 — Slliht salt and pepoer spreading to oack

3 — Definite salt and pepoer

4 — Symmetrical ;rr eying

5 •• Grey

6 — Very grey

Taole 6« C-reyneas scored of the rats at the end of
eight weeks.

be ores

Groups and)
:j?ots nos.

Males

J

4
4

1
' Fena lo s

t

' Totals

A (1-5)

! 5

: e

; i« !

4 !

' 6 i

22

B (6-10) !

! 2

• 2

\ 2 1

t 1 1

• 2 !

9

C (11-15)

! 6

3 4

i 5 !

t 1 i

\ 6

5 22

D (16-eo) !

t 2

: 2 !

' 1

! 1 1

1 8

E (21-25)

! 2

* t

• 4

t 2

! 1

{ 1 :

8

P (26-30) J

1 1

: 2

! 1

t 1 !

: 7 .

G (31-35)

i 2

J 2

• 1

: 2

! 1

i 1 i

1 8

H (36-40)

! 2

t 1

t 1 i

♦

• 1 1

i

1

r 7

Female.

£5

diets the rata were af:aln scored for preyness by the same
method ana the results are shown in Table 6»

Table 6. Oreyneas Score of the rats at the end of
12 weeks.

3c or

,'3

Groups andi
rats nost i

:*Iale

3

Females

: Totals

A (1-6) s

4

:

4

4 :

4 1

4

: 20

B (6-10) 1

1

I 2

? '.

2 J

2

: 10

C (11-15) 1

t

t 3

4

3 i

2 !

2

: 14

D (16-20)

t

1 3

2

3 :

1

S 2

: 11

B (21-25)

I 3

3

3 t

2

I 2

t 13

P (26-30)

1 3

3

3 :

2

1 2

i 13

H (31-40)

t

t 3

•
•

3

2 :

2

» 2

: 12

A comparison of Tables 5 and 6 shows that, after six
weeks on a diet containlnis pantothenic acid, the three
greyest rats in Group C had recovered from their complete
greyness and had left only the salt and pepper greyneas
exhibited by all the groups except Group A, A comparison
of rats No8# 15 and 4 receiving p-arainobenzoic acid with
Noa. 11 and 12 receiving no p-aminobenzoic acid did not in-
dicate that p-amlnobenzoic acid has any curative action on
achromotrichia. Only striking results, however, would
have been at all conclusive with the limited numbers of
rats used. The ayiariietrioally :^rey rats Hos. 4 and 12 had
not responded noticeably on either diet.

26

The other j^roups had been on their aup.lemented diets
for only four weeks. The "hlood caked" whiskers end eye
condition of rat No« 1 had been cured promptly by the low
pantothenic acid-hif;h p-aminobenzolc acid diet being fed
Group A, Tacle 6 shows that, with the exception of No, 3,
the rats in Group A were blacker than they were four weeks
previously. Rat I-Jo. 3 had developed the symmetrical grey-
ness after the addition of the pantothenic acid and p-amino-
benzoic acid to the diet.

Group A developed more symmetrical greynesa during
repigraentation than had been noticed for Group C, Another
type of symmetrical greyness was observed durin.; the re-
pigmentation. An area extending from the hips to the tail
remained grey along with the ventral part of the body after
the re^t of the body had become quite black. The partial
recovery of the rats In Group A did not support Martin's (27)
report that a low pantothenic acid-high p-amlnobenzoic acid
ratio .produced greyness.

All other groups, with the possible exception of Group
B, were definitely greyer than they had been before the
change of diets at the beginning of the ninth week. Croup
B was receiving the diet containing p-aminobenzoic acid which
Ansbacher used to cure acr.romotrichia. The slight differences
in greyness between Group B and the other groups could not
be interpreted to be due to the differeaces in diets.

There appear to be three different types of achromo-

87

trlchlaj a ooapleto greyneea prev«nt6d or cured with pan-»
tothenlc acid, a ayrrmjotrlcal greynesa prevented with adequate
pantothenic acid, but found Incurable In alx weeks with pen*
tothenic odd or pantothenic acid and p-aminobensolc acid In
the amounts used by Ansbacher, end a salt and ipepiior ^jcc eas
for which no prevention or cure was found althouf^h the tint
left for the cure oey have been too ahnrt,

Ansbacher's diet was found to produca a alight salt
and pepper greying in this study. It is possible that the
different results reported on airallar grey-hair studies
have been due to some authors noting, and sense ne^^lecting
this type 0" achromotrlchla. No hllden achromotrlchia fact-
ors were found*

SUKMAHY

1* Pantothenic acid la an Important factor In rat
•chjotaotrichla, but there are still other factors in-
volved in the prevention or cure of thla syndroae.

8« The experl?nent failed to ahow that rat achromo-
trlchia is related to the p-amlnobonzolc acid intake.

3t Male rats are more susceptible to greylnj' than
females*

4« Hnder the conditions of the experiment, nicotinio
acid and Inositol had no effect on growth*

28

ACKNOWLELGLEIiT

Appreciation la expressed to Dr. W, J, Peterson,
major Instructor, for his help In planning this study
and for his efforts toward aupplylnrr the many differ-
ent diet ingredients! to Dr. C, H. VsTiitnah for his
cooperation In the use of the small animal room and
equipmentj to Dr# Ralph M. Conrad for assistance with
the manuscriptj and to Charles A, Brownrig^ for his in-
valuaole help in the care of the rats.

29

LITERATURE CITED

(1) Anabacher, S,

Para-amlnobenzoic acid— a vitamin. Science
93ll04-lo5, 1341.

(2) Ansbachor, S,, '^isanaky, W, A., and Martin, l. J,

Para-amlnobenzoic acid and hormones, ^ed, Proc,
Part II# Fed, Amer, Soc, for Kxpt. Biol, li58,
1942,

(3) Best, C, II,, and Huntsman, M, E,

The effect of choline on tho liver fat of rata
in various states of nutrition. Jour, Phyaiol,
83 » 255-274. 1935,

(4) Corif^er, T, .V,, and Elvehjem, C« A,

The biological estimation of pyridoxine (vitamin
Bg), Jour, Biol, Ohem, 138:555-561, 1941.

(5) Dann, #, J, and Kohn, II. I.

Tho factor V (coenzyme I and II) content of rat
tissues s evidence for synthesis of nicotinic
acid by the rat. Joiir, Biol, Chem, 13o:435-442,
1940.

(6) Deuel, H, J,, Jr., Murray, 3, Hallman, L, ^^',, and

Taylor, D, B,

Ketosis, XII, The effect of choline on the keton-
urla of fastinj.; rats following a hi^^li-fat diet.
Jour, Biol, Chem. 120 « 277-283, 1937.

(7) Elvehjem, C, A., Madden, R. J,, 'ibron.j,, .. M,,

and Woolley, D. M.

Relation of nicotinic acid and nicotinic acid
amide to canine black tonp;ue. Arier, Chem, Soc,
Jour. 59:1757-1760. 1937,"

(8) Smorson, G, A.

Pailiire to cure or prevent (graying of rats with
p-aminobe azoic acid. 3oc. Expt, Biol, and Mod.
Proc. 47:448-449, 1941.

(9) Emerson, G, A,, and Evans, H, M,

Growth and graying of rats with total "filtrate
factor" ana with pantothenic acid, Soc, Fxot,
Biol, and Med, Proc. 4G: 655-658. 1941.

KANSAS STATE COLLEGZ LIBRARIES

30

(10) Engel, R, W,, and Salmon, Vv. .

Improved diets for nutritional and patholof Ic
studies OX" choline deficiency In young rats.
Jour. Nutr, 2£':10S-117. 1941,

(11) Free, A, H,

Non-identity of gray hair produced by min-
eral deficiency and vitamin deficiency, Soc,
Expt, bid. and Med, Proc, 44:371-373, 1940,

(12) Griffith, W. H,

The nutritional importance of choline. Jour,
Nutr, 2C:?39-£53. 1941,

(13) Gortor, P, J,

Dietatry depigmentation of young rats. Nature
134:382, 1934.

(14)

Depigmentation, a new dietary deficiency disease,
cured by copper. Nature 136:185, 1935,

ti

(15) Gyorgy, P,, and Poling, C, E,

Pantothenic acid and nutritional achromotrichia
In rata. Science 92:202, 1940,

(16)

i^nirther experiments on nutritional achromo-
trichia in rats and mice, Soc, Expt, Biol,
and Mod. Proc, 451773-77(5, 1940,

(17) Hartwoll, G, A.

Note on the colour changes in rats' fur pro-
duced by alterations in diet, Blochem, Jour.
17:547-546, 1923.

(18) He>r3ted, D. M,, Brigga, G. M., Mills, R. C,

iUvohjem, C, A, and Hart, E, B»

Inositol in chick nutrition, Soc, Expt, Biol,

and Med. Proc. 47:376-377, 1941,

(19) Henderson, L. M. Molntire, J. ?^., Waisman. H. A..

and Elvehjem, C. A, '

Pantothenic acid in the nutrition of the rat. Jour.
Nutr, 23:47-56, 1942,

(20) Jukes, T, H,

The pantothenic acid requirement of the chick.
Jour, Eiol, Chera, 129:225-231, 1939,

31

(21)

Eell, li, L,, and Nelson, V. K,

The role of copper in hemoglobin regeneration
and in reproduction. Jour, Eiol, Chem. 93:49-
57. 1931,

(22)

Lepkovskj', S,, Jukes, T. H«, and Krause, M. E,
The uultiple nature of the third factor of the
vitamin B complex. Jour, Biol, Chem. 115t557-
566, 1936,

(23)

LuRde, G,, and Kringstad, H,

Tiber die fiir die normal entwicklung Von Haut
und Pilz notwendigen Fsktoren im Vitamin B-
Komplex, Arhandl, Norake Videnskapa-Akad,
Oslo, I, Mat,-Naturv, Pllasse, No, i, 17 p,
1938, (Through Chem, Abs. 32: 5456-5457, 1938,)

(24)

Uber veranderunreri des pelzes von ratten durch
mangel and gewisaen factoren des vitamin E-
Komplexea, II, Z physiol, Chem, 275t20l-21b,
1939, (Through Chem. Abs, 35t3429, 1939.)

(26)

Madison, L. C,, Miller, R, C, and Keith, T, B,
Nicotinic acid in swine nutrition. Science 89j
490-491. 1932,

(26)

McElroy, L, W,, Saloman, K,, Flgge, T. H. J.,
and Cowgill, G, R,

On the porphyrin nature of the fluorescent
"blood ceked^ whiskers of pantothenic acid
deficient rats. Science 94j467, 1941,

(27)

Martin, G, J,

The achromotrichia action of p-amlnobenzoic
acid, P'ed, Proc, Part II, Fed, Amor, Soc,
for Expt, Biol, 1:58. 1942.

(28)

Martin, :, J, and Ansbacher, S,

Role of para-aminobenzoic acid in vitamin
B-complex studies with mice. Soc. Expt. Biol,
and Med. Proc. 48:118-120, 1941,

(29)

Martin, G. J,, Wlaansky, W, A,, and Ansbacher, 3.
Para-aminobenzoic acid and dopa reaction. Soc.
Expt, Biol, and Med. Proc. 47:26-28. 1941.

(30)

Morgan, A, P,, Cook, B, B., and Davison, H. G.
Vitamin Bg deficiencies as affected by dietary
carbohydrate. Jour. Nutr. 12:27-43. 1938,

32

(31) Morgan, A, P., and Sltnma, H. D,

Greying of fxir and other disturbances in
sevorni species due to a vitamin deficiency.
Jour, Nutr. 19jP33«250, 1939.

(32) Mushett, C. W,, and Unna, K.

Inefficacy of hormones in nutritional achro-
raotrichia of rata. Jour, Nutr. 2£:5o5-571, 1941,

(33) Norria, L. G. and Rln-^^rose, A, T.

The occurrence of a pellasrous-like 3yndro5ne
in chicks. Scianca 71j643, 1930.

(34) Olesoa, J. J,, Elvohjem, C. A., and Hart, • . ri.

Nutritional acbro.'notrichla. Soo. Sxpt. Biol.
and Med. Proc. 42:283-285. 1939.

(35) Pavcek, P, L, and Baum, li. M.

Inositol and spectacled eye in rata. Science
93:502, 1941,

(36) Phillips, P. H., and Hart, E, B,

The effect of orf;anic dietary constituents
upon chronic fluorine toxicosis in the rat.
Jour. Biol, Ohem, 109:657-663. 1935,

(37) Richards, R, K,

Toxicity and tolerance of para-anlnobenzoic
acid. Fed, Proc. Part II. Fed. Amer. Soc.
for Expt, Biol, 1:71-72. 1942,

(38) Rlchter, C. P. and Gllsby, K. H,

Graying of hair produced by ingestion of
phenylthiocarbamide, Soc, Exot, Biol, and
Med, Proc, 48:684-687, 1941,

(39) Rubbo, S, 1)., and Gillespie, J. M.

Para-rtminobenzoic acid as a bacterial P-rowth
factor. feature 146:838-839, 1940,

(40) Sieve, B, F,

Clinical achromotrichia. Science 94S267-25&,
1941,

(41) Spies, T, D,, Pean, W. B., and Stone, R. E.

The treatment of subclinical and classic
pellagra. Use of nicotinic acid, nicotinic
acid amide and sodi^im nicotinate, with special
reference to vasodilator action and the effect
on mental symptoms. Amer. Med. Assoc. Jour. Ill:
584-592. 1938,

33

(4?) Stekel, J. A,

On the inhibition of growth of rats by nicotinic
acid* Aba. 103rd meeting, Amer, Chem. Soc,
Memphis, Tenn. Sect. B, p. 9-10. April 20-24,
1942,

(43) Sure, B,

rietary requirements for fertility and lacta-
tion. XXX, Role of p-arainobenzoic acid and
inositol in lactation. Science 94:lti7. 1941*

(44) TJnna, K,

Pantothenic acid requirement of the rat. Jour,
Nutr. 201565-576. 1940.

(45) Unna, K,, Richards, G. V,, and Sampson, W, L,

Studies on nutritional achroraotrlchia in rats.
Jour. Hutr. 22»553-563, 1941.

(46) Vlgneaud, V, du. Chandler, J. P., '^oyer, A. W,,

and Keppel, D, M,

The effect of choline on the ability of horao-
cystine to replace methionine in the diet. Jour,
Biol. Chem. 131:57-76, 1939,

(47) Williams, R. R.

Inefficacy of pantothenic acid against the
graying of fur. Science 92 i 5 61*562, 1940.

(48) Wllllama, R, J., Truendall, J. H,, Welnstock,

H, H,, Jr., Rohrmann, E,, Lyman, C, M., and
McBur ney, C, H,

Pantothenic acid II. Its concentration and
purification from liver, Amer. Chem. Soc,
Jour, 60:2719-2723, 1938,

(49) Woods, D, D.

The relation of p»amlnobenzolc acid to the
mechanism of the action of sulphanilamide,
Brit, Jour. Expt. Path, 21:74-90, 1940,

(60) Woolley, D, W.

A new dietary essential for the mouse. Jour
Biol. Chem, 136:113-118, 1940.

(51)

Relationship of pantothenic acid and inositol
to alopecia in mice. Soc. Expt. Biol, and Med.
Proc. 46:656-669. 1941.

54

(52)

Woolley, D, W*, Waisman, H, A., and Elvehjem, C,A«
Nature and partial synthesis of ths chick anti-
dermntitls factor, Amer. Chem, Soc. Jour, 6lt
977-978, 1939.

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